A method for recovery of Candida albicans DNA from larger blood samples and its detection by polymerase chain reaction on proteinase genes.
A method for the detection of Candida albicans from up to 15 ml of blood by polymerase chain reaction (PCR), based on the differential resistance of mammalian and fungal cells towards detergent was developed. The procedure essentially involved removal of the blood cells by sodium dodecyl sulfate (SDS) induced lysis, followed by DNA extraction after degradation of fungal cell walls by a recombinant beta-1,3-glucanase. The genes of two different aspartic proteinases from C. albicans, SAP1 and SAP2, with an overall homology of 77% in their nucleotide sequences, were chosen as targets for PCR. The oligonucleotide primers used were directed to strictly conserved regions similar in both genes. As the number of base pairs between the primers are different in the two genes, amplification products of 220 bp and 238 bp in length were obtained. This led to a characteristic double band in subsequent agarose gel electrophoresis. The detection limit for a nested PCR was less than 10 C. albicans cells ml-1 of seeded blood. The detection limit of conventional PCR from a blood volume in the 10 ml range was less than 100 yeasts ml-1. Preliminary trials with clinical blood specimens suggested, that conventional PCR from large blood samples, being less laborious and prone to contamination than nested PCR, could be suited for the detection of deepseated C. albicans mycosis.
Reichard
U
U
Department of Medical Microbiology, University of Göttingen, FR Germany.
Margraf
S
S
Hube
B
B
Rüchel
R
R
D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000150:complications; D002177:Candidiasis / Q000097:blood* / Q000150:complications; D004271:DNA, Fungal / Q000097:blood*; D005260:Female; D005656:Fungal Proteins; D005800:Genes, Fungal; D006801:Humans; D007938:Leukemia / Q000150:complications; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D004271:DNA, Fungal; D005656:Fungal Proteins; D016282:Aspartic Acid Endopeptidases; C086390:SAP1 protein, Candida albicans; C090753:SAP2 protein, Candida